Griffitts:Conetainers: Difference between revisions
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* After they are done soaking, place them in the freshly cleaned [[Griffitts:Conetainers#Rack preparation|rack]] so that you can place the [[Griffitts:Conetainers#Batting square preparation|batting squares]] into the cones | * After they are done soaking, place them in the freshly cleaned [[Griffitts:Conetainers#Rack preparation|rack]] so that you can place the [[Griffitts:Conetainers#Batting square preparation|batting squares]] into the cones | ||
==Batting square preparation== | ==Batting square preparation== | ||
* Cut the white batting into 200 squares (~1 square inch each) | * Cut the white batting into 200 squares (~1 square inch each) for each conetainer being prepared | ||
* Soak them over night in a small tub with bleach | * Soak them over night in a small tub with 4 L dH<sub>2</sub>O and 130 mL bleach | ||
* Stuff them into the bottom of the | * Stuff them into the bottom of the [[Griffitts:Conetainers#Cone preparation|cones]] using sterile technique (a sterile inoculating stick is good for this) | ||
==Rack preparation== | ==Rack preparation== | ||
* Wipe down the rack well with 70% ethanol | * Wipe down the rack well with 70% ethanol |
Revision as of 11:56, 30 July 2009
Turface preparation
Note: For Best results do turface one week prior to desired planting day so that it has time to dry
- Get two large white tubs and fill them ½ full with Pro League turface
- Rinse the turface repeatedly until the water comes off clear
- Your goal is to remove the smaller particles
- Drain off all of the water and autoclave on a dry cycle for 60 minutes
- 30 min sterilize time, 30 min dry time
- For small loads, use the autoclave in 758 WIDB
- For large loads, use the autoclaves in 740 WIDB
- Remove from autoclave and immediately cover with foil to prevent contamination
- Place sterile batting squares in the cones so that turface won't fall through
- Wipe down a funnel and a scoop with 70% ethanol
- Using the sterile funnel and scoop, fill each cone to the top with turface
- Compact the turface by lifting it and pounding it against the counter
- Fill each cone again until it is about 1 cm from the top of the cone
- It won’t compact much the second time, so be sure not to overfill
- Put on gloves and spray them with 70% ethanol
- Wipe off any extra turface not in the cones
- Cover rack with Saran wrap until you are ready to plant and place dome on it
Cone preparation
- Fill the sink up with water and place 200 cones into it
- Make sure that all the roots from previous experiments have been removed—this may require scrubbing each one with the brush
- When they are all clean add bleach until you can smell it easily
- Allow them to soak for one day
- After they are done soaking, place them in the freshly cleaned rack so that you can place the batting squares into the cones
Batting square preparation
- Cut the white batting into 200 squares (~1 square inch each) for each conetainer being prepared
- Soak them over night in a small tub with 4 L dH2O and 130 mL bleach
- Stuff them into the bottom of the cones using sterile technique (a sterile inoculating stick is good for this)
Rack preparation
- Wipe down the rack well with 70% ethanol
- Using white tape, wrap it around the top of the rack so you can label each row
- Wipe down the cover with 70% ethanol
Planting
- Soak the turface well a couple times so that it doesn’t dry out
- this may take 4 L of water
- Using flamed tweezers, dig a hole in the center of each of the cones designated for each plant species
- Remove parafilm from the germinated plants and add enough dH2O to the plates to allow all the plants to be kept moist
- Using another pair of flamed tweezers plant each plant into the desired cone
- Be sure not to force the plant into the turface, so that you don’t damage the root
- Using the tweezers that are covered with turface, compact the turface around the plant
- You want the top of the cotyledon showing and that is all
- Repeat steps 4 and 5 for all the plants on the row and then steps 2–5 for all remaining rows
- After you have planted the rack cover with Saran wrap and place on the dome for a day
- On day two, when you are watering, remove Saran wrap and only use the dome from then on to cover them
Watering
Note: On average we set an experiment for 25–28 days
- During the first week, water every day with 1X SNM
- During the second week, water every other day with autoclaved dH2O
- During the third week, water every other day with 1X SNM
- During the last ½ week, water every day (except the day before you want to record your results) with dH20
Inoculation
Note: Inoculation day is four days after planting, so you will want to make sure that you have steps 1 and 2 done by Day 4 of the experiment
- Grow up all your bacterial strains to singles
- This takes 2–3 days
- From those singles patch out an ~1 cm square patch
- Fill a Falcon tube with 1 mL more of dH2O than the number of plants you will be inoculating with the same bacterial strain
- Put enough bacteria into the tube to make the solution slightly opaque
- Vortex
- Add 1 mL of the bacteria solution to each plant
- Be sure to dig the pipetter ~0.5 cm into the turface so that it doesn’t splash
- Make sure you keep track of where you are so you don’t inoculate the wrong plants)
- Repeat Steps 3–6 for all bacterial strains